The general field of the invention is the area of life sciences, particularly agricultural sciences and testing methods. Specifically, the field of the invention is the biochemistry and mode of action of Bacillus thuringiensis crystalline (Cry) endotoxins and their interactions with insect Cry toxin receptors.
Cry insecticidal proteins are endotoxins produced by Bacillus thuringiensis (Bt), a Gram positive bacterium found globally distributed in different soil types. Various classes of Cry proteins are selectively toxic against certain insect pests. The endotoxin is typically found in the form of a crystalline protein located in large inclusion bodies of the bacterium. Cry toxins have considerable sequence diversity (Crickmore et al., 1998; de Maagd et al., 2003), but the majority of the toxins that have activity against lepidopteran pests are 130 kDa protoxins having a three domain active core toxin structure (de Maagd et al., supra.).
The subject of the current invention relates to the Cry1Fa toxin, a three domain Cry protein. This toxin has demonstrated insecticidal activity against various lepidopteran insects, including Spodoptera frugiperda (J. E. Smith) (fall armyworm) and Ostrinia nubilalis (Hübner) (European corn borer), which are two of the most economically important insect pests of maize. Cry1Fa is the toxin component of two USDA deregulated transgenic plant incorporated pesticides know as event TC1507 in maize (HERCULEX®) and event 281-24-236 in cotton (WIDESTRIKE®).
Cry1Fa full-length holotoxin protein, like other three-domain Cry toxins, requires proteolytic cleavage at both the N-terminus and the C-terminus ends for activation of its insecticidal activity. The midguts of lepidopteran insects contain a variety of trypsin and chymotrypsin-like proteases that process the full length holotoxin to a core toxin structure having a size of approximately 68 kDa (Christeller et al., 1992; Gatehouse et al., 1997; Bernardi et al., 1996). The processing involves removal of approximately 28 amino acids from the N-terminus and approximately 530 amino acids from the C-terminus (protoxin segment), and the resulting core toxin segment is released and binds to specific receptors located within the insect gut.
Insects can develop resistance to the activity of Cry protein toxins through changes in midgut localized receptors that bind the Cry protein core toxin (Heckel et al., 2007; Van Rie et al., 1990b). Further, other mechanisms of resistance development have been documented, including: reduced activation of the protoxin, changes in the number of Cry receptors in the insect midgut, and the loss of the ability to respond to the toxin by formation of membrane pores that contribute to insect mortality (see Griffitts and Aroian, 2005; and Van Rie et al., 1990b).
Prior to this invention, studies to characterize the binding of Cry1Fa core toxin protein to different insect receptors have not been reported. The reason being was that traditional radio-labeling methods involving oxidized iodine isotopes reacting with tyrosine residues in the Cry1Fa toxin produced radiolabeled core toxin protein that lost its ability to bind to receptors in brush border membrane vesicles (BBMVs) prepared from midguts of Spodoptera exigua and Spodoptera frugiperda (Luo et al., 1999). Further, it was found that traditionally radiolabeled Cry1Fa core toxin protein lost its insecticidal activity and was inactive in diet bioassay's against these Spodoptera species. Other methods of protein labeling such as fluorescent labeling, and other methods to measure ligand-receptor binding such as isothermal calorimetry, have been attempted, but have been found to be either too insensitive, or the optical methods too difficult to use due to the particulate properties of insect BBMVs.
Palmer et al. (1997) described an indirect method of radioactive labeling of proteins specifically at cysteine residues. In this method, an intermediate compound, fluorescein-5-maleimide, is first reacted with radio-iodine, then the radiolabeled fluorescein-5-maleimide is used to chemically modify the protein at available cysteine residues. This invention describes the use of the highly specific Palmer et al. method to radiolabel Cry1Fa protein by targeting a single cysteine residue (C205) located in Domain 1 of the Cry1Fa core toxin.
It was most surprising to find that the introduction of this stearically cumbersome radiolabeled 5-maleimide into the Cry1Fa core toxin protein prepared by this method did not result in a loss of binding to its receptor or cause toxin inactivation. As a result, this non-traditionally radiolabeled Cry1Fa core toxin maintained sufficient tertiary protein structure to retain both its insecticidal activity and its ability to bind specifically to receptors in BBMV preparations from a variety of insects.
The non-traditionally radiolabeled Cry1Fa protein was found to bind to receptors in a saturable manner, and was used in a competitive binding assay to determine if other Cry toxins compete with its binding. Using this assay it was demonstrated that field resistance to Cry1Fa toxin that developed in a population of S. frugiperda collected in Puerto Rico is due to the loss of the ability of receptors in BBMVs of these insects to bind Cry1Fa core toxin protein.